笔者想涡移植的宝贵意见，以及过去和现在的桑切斯实验室成员感谢千代子小林和Kiyokazu阿加塔对这些技术的发展过程中的宝贵讨论。这项工作是由美国国立卫生研究院训练津贴（5T32 HD0791）主计长办公室和美国国立卫生研究院R37GM057260亚撒支持。 ASA是霍华德·休斯医学研究所的研究员。

注:此协议表明，潜在的有害物质的使用（铅和chloretone的）。收购，读，并按照对所有潜在的有害物质的MSDS。 1。动物文化，选择和制备<ol><li>动物养殖和处理使用涡水（1X 9 Monjuic蒙特惠奇盐）和塑料转让移液器。 </li><li>粉虱Schmidtea地中海性可用于在实验室中提出，在正常培养条件10。无性标本到生产所需的大小， 与地中海提出在正常情况下，在室温下，应投喂在使用前一至两个月的一倍至三倍的正常频率（每周2-3次）。另外，在正常频率无性动物喂食，可保持在10度摄氏无限期，以增加他们的平均规模。 </li><li>选择1至2厘米之间的动物长度和宽度超过2毫米，然后饿死的动物3-7天前使用。 </li><li>如果目的主机上，捐助者，或局部照射动物进行任何药物或放射治疗，在这一点上执行的处理（S）。 </li><li>如果进行药物治疗，需要喂养的动物，饿死动物额外的3至7天前使用。 </li></ol> 2。解决方案和材料的制备<ol><li>涡水溶解在0.1-0.2％W / V chloretone和寒心的解决方案上的冰准备，轻度局部麻醉剂，chloretone解决方案。 </li><li>如果进行局部照射，继续加强2.7。组织移植，继续加强2.3。 </li><li>使用本生灯，0.75 mm内部直径，用来切割移植组织，和0.7毫米外径，用于创建一个主机将收到1-2厘米的移植，毛细管90°角的孔弯曲从EA的结束CH管。为了节省材料，每个毛细管两端弯曲，打破他们的一半产生两个工具。小心不要管非常两端火焰。 </li><li>削减到指定大小的下列文件: <ul><li>黑色滤纸（切成长方形约2.5厘米×1.5厘米） </li><li>滤纸＃3滤纸（切成长方形约2厘米×0.5厘米） </li><li> kimwipe（折叠切成WADS约。3厘米×0.5厘米×4层） </li><li>卷烟纸（清除口香糖的条状，切成长方形约3厘米×2厘米） </li></ul></li><li>准备修改Holtfreter的解决方案（3.5克/ L的NaCl，0.2 g / L的碳酸氢钠 ，0.05 g / L的氯化钾，0.2 g / L的硫酸镁 ，0.1 g / L的氯化钙 ，pH值7.0-7.5）和酪蛋白饱和Holtfreter的解决方案和寒冷同时4°C。 </li><li>将折叠Kimwipe佩尔蒂埃冷却器板或其它冷却设备，坐落在解剖显微镜下的封口膜广场和地方。饱和的与冰鲜Holtf Kimwipereter的解决方案和地方两个黑色的Kimwipe滤纸矩形。 </li><li>线滤纸＃2滤纸的培养皿。浸湿的滤纸与Holtfreter的解决方案和冷藏冰菜肴。一个更大的盘子和滤纸班轮局部照射也可使用。 </li></ol> 3。麻醉和固定<ol><li>填写入菜与冰鲜chloretone解决方案和吸管虫的培养皿。仅用于移植的麻醉一台主机和一个捐助一次。许多动物（N&gt; 10），局部照射可以是麻醉一次。 </li><li>允许蠕虫，直到他们成为一动不动在chloretone溶液浸泡（5-10分钟）。 </li><li>冲洗吸取到他们与冰鲜Holtfreter的解决方案，填补了菜虫。 </li><li>吸取到黑色过滤纸浸透与冰鲜Holtfreter的解决方案和东方钳腹朝下固定动物。如果动物能够禄表参，吸收多余Holtfreter的解决方案，略有下降，您的佩尔蒂埃或冷板的温度，或增加chloretone治疗的长度。 </li></ol> 4。局部照射注意:请按照下列步骤准备动物局部照射。如果进行移植，而不是进行到第5。 <ol><li>从步骤2.7冰在冰桶内的顶级源X-射线照射，将适合放置冷冻培养皿。 </li><li>培养皿中的麻醉动物移动的黑色的滤纸上，他们是固定的安排。使用钳直接移动麻醉蠕虫，可能会伤害他们。 </li><li>运输安排顶级源X-射线照射的动物和位于从阴极管动物的距离最小，冰桶等，从而最大限度地有效剂量率。 </li><li>位置铅屏蔽（S）（ 图1）之间的动物和阴极ţUBE需要。盾牌应该是4.5到6毫米厚，以便为97至99 1 325kV X射线束的11％衰减。 </li><li>提供X射线剂量。如果需要完整的非屏蔽地区的干细胞消融，提供30 Gy或使用的X-射线照射。供参考，是30戈瑞相当于3.6分钟，在320千伏和10毫安，在以场源的距离为30厘米的精密X射线公司XRAD320。 </li><li>立即服药后完成后，处理的黑色滤纸，转让动物蠕虫进入冷涡水。允许涡水升温至室温和动物逐出自己的黑色滤纸。局部照射程序现已完成。 </li></ol> 5。组织移植<ol><li>使用移液管，镊子，安排单独的矩形黑色滤纸上Kimwipe麻醉主机和捐助者的蠕虫已冷却的PeltiER或冷却器板的解剖显微镜下。 </li><li>使用0.75毫米内径的毛细管从供体切出移植插件，使用镊子，放置在了主机的方式部分。如果移植材料在毛细管被卡住了，打跑钳。 </li><li>使用0.7毫米外径毛细管从主机中取出一个插件，使用钳位置移植到留下的洞。 </li><li>转让其黑到在步骤2.7准备的培养皿滤纸矩形移植主机。 </li><li>卷烟纸湿了一块酪蛋白饱和Holtfreter的解决方案框图如下图2A，并把它移植主机上。 </li><li>浸泡在酪蛋白饱和Holtfreter的解决方案和四片滤纸包住移植主机框图如下图2B。 </li><li>四切Kimwipe WADS浸泡在酪蛋白饱和Holtfreter的解决方案，并奠定他们步骤5.6滤纸（ 图2B）。替换盖和冰放在培养皿。 </li><li>转移到涡水，恢复，愈合和再生捐助的蠕虫病毒。 </li><li>当所有移植完成后，将移植到10℃培养箱蠕虫过夜。 </li><li>翌日清晨，照顾不来打扰的移植，发现蠕虫和转让（黑色滤纸）培养皿中充满了涡虫水。 </li><li>允许蠕虫逐出滤纸本身或用钳子轻轻取出。 </li><li>一次改变涡水2-3天。 </li></ol> 6。代表结果紧接着会出现局部照射涡虫正常，不受影响。根据交付的剂量和使用屏蔽的几何形状，照射组织可能倒退甚至解体7。屏蔽组织应保持不变。 FOLL由于组织回归和组织完整性的损失，芽将形成和失踪结构的再生（ 图3A）。如果截肢的部分照射的区域，被照射的组织将被救出（即防止倒退或瓦解）（ 图3B）。在健侧和截肢的情况下再生相比，被切除的非辐照涡虫（ 图3C），将被推迟。如果X射线剂量30 Gy的交付和部分照射的动物是无恙，成功的干细胞可以被确认使用的铅屏蔽相应的2至3天，局部照射后原位杂交技术，干细胞的模式消融标记SMED PIWI-1 12（又名smedwi-1）（ 图4）。 移植后的早晨，一个成功的嫁接移植的组织应该是显而易见的瓦特ithin宿主组织，加入到主机（ 图5A）的背部和腹部表面。偶尔，移植将坚持只腹侧或背侧表面。如果移植是彻底失败的，没有移植的迹象将是从主机（ 图5B）背或腹面可见。不久后的非辐照组织的成功移植到已经消融的干细胞原位杂交致死量照射13,14，SMED PIWI-1，就会发现主要在移植干细胞存在（ 图5C主机）。此外，非辐照组织的成功移植到致死剂量照射的主机将导致宿主组织的救援和长期生存的主机15。 <img alt="图1" src="/files/ftp_upload/4015/4015fig1.jpg" /> 图1。大会安排的基本组件在顶级定位的X射线源的X射线照射局部照射。（阴极管）麻醉涡位置下方的X射线源直接照射野内。为了最大限度地利用X射线剂量率，涡虫和X射线源之间的距离应尽量减少。铅屏蔽应定位之间的阴极管及麻醉蠕虫，尽可能接近实际的蠕虫。铅屏蔽应设计，制造和定位，所以它盾牌所需的组织，但完全暴露了其余的蠕虫。许多商业生产将产生定制的铅屏蔽，从简单的设计图，我们已经成功地使用了Alpha系统公司（Bluffdale，UT斯达康）。铅应该足够厚的屏蔽，以便需要的金额。例如，如果需要接近完成一个320千伏的X射线束屏蔽，铅应是4.5至6毫米厚的。涡虫和盾牌定位1 Holtfreter浸泡滤纸的培养皿中冰桶在于内衬。给出一个足够大的X射线辐射场和一些相同的铅屏蔽，许多标本可部分照射一次（图）。 <img alt="图2" src="/files/ftp_upload/4015/4015fig2.jpg" /> 图2。建设恢复室 （一）组织移植恢复室爆炸视图，显示所有被浸泡在酪蛋白饱和Holtfreter的解决方案后，顶部彼此分层组件。 （二）一个接近完成恢复室，说明环环相扣的滤纸安置＃3滤纸矩形紧贴包住麻醉涡，防止愈合过程中的运动。恢复室精心施工，防止动物运动和干燥，促进快速愈合和组织移植的更大的功效。 <img alt="图3" src="/files/ftp_upload/4015/4015fig3.jpg" /> 图3。代表成果简单后局部照射。杜波依斯描述7，（a）当涡虫的后半屏蔽铅，然后暴露在X射线照射前组织观察倒退回边界之间的辐射和屏蔽组织在这点未染色blastemas形成和动物开始再生。 （二）另一方面，当动物被斩首后，执行相同的局部照射（一），组织回归没有观察到，其余辐照前组织被救出。断头部分照射的动物再生头（二），然而，再生了显著延迟比未辐照（三）断头控制。 <img alt="图4" src="/files/ftp_upload/4015/4015fig4.jpg" /> 图4。代表outco我偏干细胞消融后局部照射。SMED PIWI-1的干细胞标记的原位杂交（WISH）全安装显示，野生型涡虫干细胞分布整个身体组织的异常与前感光细胞（箭头）和适当的咽（星号）14。 （一）希望SMED PIWI-1照射，但完全屏蔽控制涡虫固定3天照射后显示了干细胞的分布，是区分野生型涡虫。 （二）另一方面，希望为动物，只有部分屏蔽SMED PIWI-1，离开的前部和后部暴露，但也固定三天后照射表明，干细胞是从非屏蔽地区消融。比例尺是500微米。 <img alt="图5" src="/files/ftp_upload/4015/4015fig5.jpg" /> 图5。 EXA组织移植成功和不成功的mples。（一）实时图像成功移植的涡三天后移植的背，腹意见。移植（指示）清晰可见，背部和腹部表面，并与未染色的特点组织包围在移植物抗宿主接口。 （二）相应地，不成功移植显示不可见移植组织在移植网站（指示），而显示，从失败的移植愈合的，未染色的侧切伤口。只背或腹面坚持一个移植，可以像（一）从一个侧面和一个不成功的移植（二）从其他成功的移植。 （C）当野生型（WT）组织居民干细胞移植的干细胞已被消融到照射主机嫁接以后显露希望SMED PIWI-1天移植后，移植的成功清楚地显示干细胞移植物的位置（箭头）或周围的具体存在。比例尺是500微米。

<ol>
	<li>Morgan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+the+regeneration+of+Planaria+maculata.">Experimental studies of the regeneration of Planaria maculata.</a> Arch. Entw. Mech. Org. 7, 364-397 .</li><li>Reddien, P. W., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+planarian+regeneration.">Fundamentals of planarian regeneration.</a> Annu Rev Cell Dev Biol. 20, 725-757 (2004).</li><li>Randolph, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regeneration+of+the+tail+in+lumbriculus.">The regeneration of the tail in lumbriculus.</a> J. Morphol. 7, 317-344 .</li><li>Wagner, D. E., Wang, I. E., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+neoblasts+are+pluripotent+adult+stem+cells+that+underlie+planarian+regeneration.">Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.</a> Science. 332, 811-816 (2011).</li><li>Santos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transplantation+in+planaria.">Studies on transplantation in planaria.</a> Biological Bulletin. 57, 188-197 (1929).</li><li>Morgan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+grafted+pieces+of+planarians.">Regeneration of grafted pieces of planarians.</a> J. Exp. Zo&#246;l. 3, 269-294 (1906).</li><li>Dubois, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+&#225;+l+'&#232;tude+de+la+migration+des+cellules+de+règènèration+chez+les+Planaires+dulcicoles.">Contribution &#225; l '&#232;tude de la migration des cellules de règènèration chez les Planaires dulcicoles.</a> Bull. Biol. Fr. Belg. 83, 213-283 (1949).</li><li>Purton, L. E., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell Stem Cell. 1, 263-270 (2007).</li><li>Cebria, F., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+homologs+of+netrin+and+netrin+receptor+are+required+for+proper+regeneration+of+the+central+nervous+system+and+the+maintenance+of+nervous+system+architecture.">Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture.</a> Development. , 132-3691 (2005).</li><li>Newmark, P. A., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+specifically+labels+the+regenerative+stem+cells+of+planarians.">Bromodeoxyuridine specifically labels the regenerative stem cells of planarians.</a> Dev. Biol. 220, 142-153 (2000).</li><li>Miller, W., Kennedy, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+attenuation+in+lead,+aluminum,+and+concrete+in+the+range+275+to+525+kilovolts.">X-ray attenuation in lead, aluminum, and concrete in the range 275 to 525 kilovolts.</a> Radiology. 65, 920-925 (1955).</li><li>Pearson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-based+whole-mount+in+situ+hybridization+method+for+planarians.">Formaldehyde-based whole-mount in situ hybridization method for planarians.</a> Dev. Dyn. 238, 443-450 (2009).</li><li>Hayashi, T., Asami, M., Higuchi, S., Shibata, N., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+planarian+X-ray-sensitive+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting.</a> Dev. Growth Differ. 48, 371-380 (2006).</li><li>Reddien, P. W., Oviedo, N. J., Jennings, J. R., Jenkin, J. C., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMEDWI-2+is+a+PIWI-like+protein+that+regulates+planarian+stem+cells.">SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells.</a> Science. 310, 1327-1330 (2005).</li><li>St&#233;phan-Dubois, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Les+cellules+de+r&#233;g&#233;n&#233;ration+chez+la+planaire+Dendrocoleum+lacteum.">Les cellules de r&#233;g&#233;n&#233;ration chez la planaire Dendrocoleum lacteum.</a> Bulletin de la Société Zooologique de France. 86, 172-185 (1961).</li><li>Gurley, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+secreted+Wnt+pathway+components+reveals+unexpected+complexity+of+the+planarian+amputation+response.">Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response.</a> Dev. Biol. 347, 24-39 (2010).</li><li>Salvetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+stem+cell+plasticity:+neoblast+repopulation+in+non-lethally+irradiated+planarians.">Adult stem cell plasticity: neoblast repopulation in non-lethally irradiated planarians.</a> Dev. Biol. 328, 305-314 (2009).</li></ol>

没有利益冲突的声明。

固定化的重要性固定是迄今为止对这些过程的任何成功完成最关键的一步。如果涡虫是不当的固定部分照射之前，他们可能将下方的铅屏蔽，产生不一致的，混杂的结果。此外，如果动物没有得到充分固定移植后，主机的蠕虫病毒可能会搬走的移植组织，导致移植失败，在妥善医治主机。适当的固定是通过调整浓度或chloretone治疗的长度和滤纸的温度和湿度对涡虫休息。然而，在长达一个半小时的0.2％chloretone治疗不会出现有毒，处理，长度可能诱发咽弹射。涡虫仍保持在这段时间的长度和服从局部照射。我们的经验表明，水分，而不是chloretone治疗，是最关键的因素 - 太湿和动物移动方便，过于干燥和动物将变干。在所有情况下，滤纸应完全饱和，但不能创建积水容器内，或表面上的点。多余的液体可以从培养皿动摇。的液体使用量饱和的滤纸将毫无疑问，需要根据具体的环境湿度进行调整。 移植的速度速度是增加了两个部分的照射和移植的疗效至关重要。较长的部分照射的动物固定受伤的可能性越大或组织的完整性臀位可能歪斜的一个特定的检测结果。移植是预制的速度大致与成功接枝率。如果是接受移植的主机洞空时间过长，受伤的腹侧和背侧表面会开始愈合，而不是互相移植组织，从而导致不​​成功的移植。在我们的手中，移植插件只是偶尔变得在毛细管提出。插件可以在这些场合，通常被抛下，迅速用细镊子或通过迫使空气通过毛细管口。一旦成为实行移植手术，所需的时间产生许多嫁接的动物，可大大减少由麻醉，同时嫁接前对未来的捐助主机对。实际所需的时间生产各种嫁接的动物进行麻醉和移植，同时，可以下降到低至5分钟。 局部照射和移植的多功能性一旦麻醉和局部照射的条件进行了优化，根据具体的实验environmeNT和可用的X射线照射，许多不同的实验，可以进行局部照射技术变大。铅加工技术，让先进的生产不仅错综复杂的照射，可以针对特定器官和组织的盾牌，但也制造大量相同的盾牌，允许快速建立的许多生物复制。毫无疑问，今后的调查将采取这种方法的优点，以测试在体内的细胞反应，局部干细胞消融，目前在涡虫的不同部位的干细胞群之间的功能差异。局部照射的多功能性，因为它涉及到科学问题的兴趣，只限于电流引线的加工技术和人的想象力。因此，局部照射技术的真正威力在于能够精确地屏蔽和消融几乎任何不同的定位对干细胞的亚群。 而移植干细胞亚群的隔离允许，这种技术更重要的优势是差异对待的主机或捐助者移植前的能力。治疗药物或RNA干扰与主机或捐助，将允许未经处理的移植细胞的行为如何处理环境或未经处理的环境行为如何处理的细胞中的调查。这些类型的实验，有一定的帮助捉弄除了自治和非自治分子捐款，干细胞的功能。 此外，作为经典的嫁接在涡虫的实验已经显示出了5,6，组织移植是一个理想的技术，探索不同组织的再生过程中的感性潜力。由于涡虫的研究进入了分子时代，我们已经开始发现IMP的本地化非常具体的表达ortant信号分子直接控制再生16。移植组织包含这些信号分子在再生过程中的异位网站，可以帮助我们更好地了解他们的作用，引导再生结构的形态。 局部照射和组织移植的优势超过其他技术除了固有的特定优势，局部照射和组织移植相比，到另一个时，他们还提供了涡虫的再生领域中使用的其他技术具有独特的优势。例如，低剂量辐射已被利用作为一种隔离的少数干细胞内，否则干细胞缺乏主机4,17，但是，因为我们不知道如何亚致死剂量照射影响涡虫干细胞，我们不能肯定在这些实验中观察到的细胞行为是典型的正常。这种低剂量照射的警告是在局部照射的关注，因为辐射照射可以很容易衰减，适当厚度的铅超过​​99％。此外，移植完全可以避免这个警告，因为人口少的干细胞可以分离出干细胞缺乏主机内没有暴露移植的细胞对辐射。此外，健康的干细胞的确切位置，可以了解和控制在局部照射和移植，而低剂量辐射的细胞存活随机定位在整个动物。最后，而单 ​​细胞移植优雅展示了一个单一的涡虫干细胞的高潜力，组织移植可证明一个同样强大的技术，因为它是更快，少乏味，同时可以观察到细胞的功能和行为在每个主机。人口少的细胞分析对单个细胞不仅可以更有效地收集数据，但它也可能揭示细胞与细胞的相互作用，在一个单一的细胞移植法将错过。 结论局部照射和组织移植，虽然旧的技术，在解剖涡虫干细胞的功能，以及一般的基本再生的分子机制将发挥重要的作用。这些传统技术的现代化带来了更高的吞吐量和更大的一致性，同时允许为现代功能的检测和分子生物学技术的集成。这些曾经被用来勾勒涡虫再生的重大问题的经典技术，现在可以用来发现这些问题的答案，因此我们的干细胞生物学的理解。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>通用移液管</td><td>萨姆肯</td><td> 691 </td></tr><tr><td>毛细管（编号0.75毫米） </td><td>金控</td><td> 30-30-0 </td></tr><tr><td>毛细管（外径0.7毫米） </td><td>金控</td><td> 30-50-08 </td></tr><tr><td>封口膜中号</td><td>厂商VWR </td><td> 52858-076 </td></tr><tr><td> kimwipes 34155 </td><td>厂商VWR </td><td> 500029-891 </td></tr><tr><td>黑色滤纸</td><td>施莱歇尔＆Schuell </td><td> 10310809 </td></tr><tr><td>滤纸＃2过滤纸1002-055 </td><td> Fisher Scientific则</td><td> 09-810B </td></tr><tr><td>滤纸＃3过滤纸1003-185 </td><td> Fisher Scientific则</td><td> 09-820E </td></tr><tr><td>卷烟纸</td><td>锯齿形，原</td><td>不适用</td></tr><tr><td> Petri网菜</td><td>厂商VWR </td><td> 82050-544 </td></tr><tr><td>钳杜蒙，Inox公司＃5 </td><td>福斯特</td><td> 11251-20 </td></tr><tr><td> chloretone </td><td> Sigma Aldrich公司</td><td> 112054 </td></tr><tr><td>酪蛋白</td><td> Sigma Aldrich公司</td><td> C3400 </td></tr><tr><td>铅屏障</td><td> Alpha系统，UT斯达康公司，Bluffdale </td><td>定制设计</td></tr><tr><td> XRAD-320生物照射</td><td>精密的X射线，北布兰福德，CT </td><td>不适用</td></tr></tbody></table>

planarian immobilization, partial irradiation, and tissue transplantation

涡虫，淡水涡虫，已被证明是一个功能强大的系统解剖的后生再生和干细胞生物学1,2。涡虫再生的任何丢失或损坏的组织，是由成人干细胞可能称为neoblasts 3。虽然已明确显示，这些干细胞是多能干细胞和奇能够再造一个完整的动物4，在干细胞的细胞行为的人口和动态异质性在很大程度上仍然悬而未决。由于大量干细胞广泛分布在整个涡虫的身体计划，需要操纵分子和功能的研究在体内的干细胞亚群的先进方法。 组织移植和局部照射两种方法，可以为进一步研究分离涡虫干细胞亚群。每种技术都有明显的优势。 ţ问题移植允许引进的干细胞，成一个天真的主机，是天生的基因不同，或此前已治疗药物。另外，局部照射使干细胞内的一台主机，并列组织的干细胞缺乏的隔离，没有伤口的引进，臀位或任何组织的完整性。使用这两种方法，一个可以探讨的细胞自主和非自主因素，控制干细胞的功能，如增殖，分化和迁移。 5,6两个组织移植和局部照射7已被用于定义许多正在研究的问题，今天依然对涡虫再生的历史。然而，这些技术仍然未得到充分利用，由于以前的方法费时费力，不一致的性质。这里提出的协议，代表一个大的进步，在减少时间ND努力要再现嫁接或部分照射的动物产生大的人数接近100％的功效。我们涵盖文化的大型动物，固定化，局部照射，组织移植的准备，和动物复苏的优化。此外，这里所描述的工作表明局部照射方法为使用最广泛研究的涡，Schmidtea地中海的首次应用。此外，高效的组织移植在涡虫，打开门的功能测试天真或处理的干细胞亚群在复育实验，这早已是金标准方法化验成人干细胞的潜力在哺乳动物8。这些技术的广泛采用，无疑会导致更好地了解的组织稳态和再生过程中的成体干细胞的细胞行为。

Watch this Scientific Journal Video about Planarian Immobilization, Partial Irradiation, and Tissue Transplantation at JoVE.com

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

一个有效的方法是明确和一贯的涡虫之间的大小为嫁接组织描述。还包括如何结合局部照射活体动物，铅屏蔽的固定化技术用于移植，可适应的描述。

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

planarian-immobilization-partial-irradiation-tissue

Nanyang Technological University

Research

JoVE Journal

Biology

19.5K 次观看.  University of Utah School of Medicine. 一个有效的方法是明确和一贯的涡虫之间的大小为嫁接组织描述。还包括如何结合局部照射活体动物，铅屏蔽的固定化技术用于移植，可适应的描述。

视频: Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

原位大鼠后肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

科研

免疫与感染

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

生物工程

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

癌症研究

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

发育生物学

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

生物学

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

利用外束辐射对小型动物模型的多能干细胞衍生畸胎瘤进行靶向选择性治疗

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

医学

Partial Heterotopic Hindlimb Transplantation Model in Rats

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

大鼠部分异位欣德利姆移植模型

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

改良异位异体下肢骨皮瓣模型在大鼠转化血管化复合同种异体移植研究中的应用

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

采取下一步:神经整形正交后肢移植模型，以最大限度地提高大鼠的功能恢复

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...